Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus.

An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.

Folding, unfolding, and refolding of the vesicular stomatitis virus glycoprotein.

Folding and refolding of the vesicular stomatitis virus (VSV) glycoprotein (G protein), New Jersey serotype, were studied both in infected cells and after urea denaturation and reduction of isolated protein in vitro. To assess the contribution of disulfide bonds to the conformation of this type I membrane glycoprotein, reduced and alkylated forms were compared with unreduced G proteins by their mobility on SDS-polyacrylamide gels and by their reactivity with conformation-dependent monoclonal antibodies (MAbs). Pulse-chase experiments showed that G protein folding in the endoplasmic reticulum (ER) of infected cells occurred rapidly (estimated half-time of 1-2 min) and involved transient association with the ER chaperone calnexin. Inhibition of glycosylation by tunicamycin slowed the folding process and emergence from the ER but did not prevent the appearance of a conformationally mature transport-competent G protein. For in vitro refolding studies, native G protein isolated from virus particles was denatured and reduced with urea and beta-mercaptoethanol. When rapidly diluted into a denaturant-free buffer containing oxidized glutathione and the nonionic detergent octyl glucoside, the G protein regained considerable native structure, as determined by reactivity with five monoclonal antibodies specific for different conformation-dependent epitopes. Whereas the refolding process was slow and inefficient in vitro relative to folding in the cell, this observation nonetheless demonstrated that an integral fully glycosylated membrane protein can be refolded to form a structure similar to that of the original protein processed during in vivo synthesis. If, however, unfolded nonglycosylated G protein was the starting material, refolding in vitro failed. In summary, we have shown that VSV G protein folding can be analyzed both in vivo and in vitro and that folding in the cell involves at least one chaperone and can occur in vivo even if not glycosylated.

Low synonymous site variation at the lacY locus in Escherichia coli suggests the action of positive selection.

We have determined the nucleotide sequences of seven lacY alleles isolated from natural isolates of Escherichia coli. Nucleotide heterozygosity estimates for this locus were compared to those obtained from previous studies of intraspecific variation at chromosomal loci, revealing that lacY has unusually low synonymous site variation. The average pairwise heterozygosity of synonymous sites (Ks = 0.0112 +/- 0.0100) is the second lowest reported and the lowest for loci that have an equivalent level of nonsynonymous variation. We consider several hypotheses to explain how different forces in evolution could act to create the observed pattern of polymorphism, including selection for translational efficiency and positive selection. Our analysis most strongly supports the hypothesis that positive selection has acted on the lacY locus in E. coli.

Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus.

Transfection of mammalian CV1 cells with a recombinant M-gene pTM1 plasmid, driven by vaccinia virus-expressed phage T7 polymerase, resulted in the expression of matrix (M) protein, which is progressively released from the exterior surface of the transfected-cell plasma membrane. Exocytosis of M protein begins 2 to 4 h posttransfection and reaches a peak by 10 to 16 h posttransfection; dye uptake studies reveal that > 97% of cells are alive and have intact membranes at 16 h posttransfection. Density gradient centrifugation and labeling with radioactive palmitic acid revealed that the M protein is released from cells in association with lipid vesicles. Expression of M-gene deletion mutants suggests that exocytosis of M protein requires the presence of a membrane-binding site at N-terminal amino acids 1 to 50. Cells transfected with the pTM1 plasmid containing the M gene of the temperature-sensitive mutant tsO23 expressed ample quantities of the mutant M protein at permissive (31 degrees C) and restrictive (39 degrees C) temperatures, but the exocytosis of the mutant M protein occurred only at the permissive temperature. The tsO23 M gene has three site-specific mutations resulting in amino acid substitutions at residues 21, 111, and 227. Expression of wild-type and mutant M genes with mutations or revertants at each of these sites resulted in exocytosis of M protein at the nonpermissive temperature only when wild-type leucine was present at residue 111, but M-protein exocytosis was restricted (to some extent even at the permissive temperature) when mutant phenylalanine was present at residue 111. Past and present data indicate that a specific structural conformation of the M protein is responsible for the formation and budding of vesicles, a property of the M protein which probably also promotes vesicular stomatitis virus assembly and budding of virions from host cells.

Nucleus-targeting domain of the matrix protein (M1) of influenza virus.

The matrix protein M1 of influenza virus A/WSN/33 was shown by immunofluorescent staining to be transported into the nuclei of transfected cells without requiring other viral proteins. We postulated the existence of a potential signal sequence at amino acids 101 to 105 (RKLKR) that is required for nuclear localization of the M1 protein. When CV1 cells were transfected with recombinant vectors expressing the entire M1 protein (amino acids 1 to 252) or just the first 112 N-terminal amino acids, both the complete M1 protein and the truncated M1 protein were transported to the nucleus. In contrast, expression in CV1 cells of vectors coding for M1 proteins with deletions from amino acids 77 to 202 or amino acids 1 to 134 resulted only in cytoplasmic immunofluorescent staining of these truncated M1 proteins without protein being transported to the nucleus. Moreover, no nuclear membrane translocation occurred when CV1 cells were transfected with recombinant vectors expressing M1 proteins with deletions of amino acids 101 to 105 or with substitution at amino acids 101 to 105 of SNLNS for RKLKR. Furthermore, a synthetic oligopeptide corresponding to M1 protein amino acids 90 to 108 was also transported into isolated nuclei derived from CV1 cells, whereas oligopeptides corresponding to amino acid sequences 25 to 40, 67 to 81, and 135 to 164 were not transported into the isolated cell nuclei. These data suggest that the amino acid sequence 101RKLKR105 is the nuclear localization signal of the M1 protein.

Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus.

The membrane-binding affinity of the matrix (M) protein of vesicular stomatitis virus (VSV) was examined by comparing the cellular distribution of wild-type (wt) virus M protein with that of temperature-sensitive (ts) and deletion mutants probed by indirect fluorescent-antibody staining and fractionation of infected or plasmid-transfected CV1 cells. The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive temperature; wt VSV also causes rounding, which prohibits study of M protein distribution by fluorescent-antibody staining. Little or no M protein can be detected in the plasma membrane of cells infected with tsO23 at the nonpermissive temperature, whereas approximately 20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 at the permissive temperature. Cells transfected with a plasmid expressing intact 229-amino-acid wt M protein (M1-229) exhibited cytopathic cell rounding and actin filament dissolution, whereas cells retained normal polygonal morphology and actin filaments when transfected with plasmids expressing M proteins truncated to the first 74 N-terminal amino acids (M1-74) or deleted of the first 50 amino acids (M51-229) or amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were readily detectable in the plasma membrane and cytosol of transfected cells as determined by both fluorescent-antibody staining and cell fractionation, as was the plasmid-expressed intact wt M protein. However, the expressed doubly deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionation, suggesting the presence of two membrane-binding sites spanning the region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in vitro binding assay in which intact M protein and its deletion mutants were reconstituted in high- or low-ionic-strength buffers with synthetic membranes in the form of sonicated unilammelar vesicles. The results of these experiments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membrane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.

Common distribution of antigenic determinants and complementation activity on matrix proteins of two vesicular stomatitis virus serotypes.

To compare the antigenic and functional domains of the matrix (M) proteins of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ), deletion mutants and chimeras were cloned in pBSM13 and expressed as in-frame lacZ fusion proteins in Escherichia coli. Non-cross-reactive monoclonal antibodies directed to the two antigenically distinct M proteins were tested by Western blot analysis to map three epitopes of VSV-Ind M protein and four epitopes of VSV-NJ M protein. Epitope 1 of the VSV-Ind M protein and epitope II of the VSV-NJ M protein both mapped to the highly basic N-terminal 34 amino acids of each homotypic M protein. Epitopes 2 and 3 of the VSV-Ind M protein and epitopes III and IV of the VSV-NJ M protein mapped to a region spanning amino acids 35 to 74. Epitope I of the VSV-NJ M protein mapped to a region between amino acid 75 and the C terminus. The similarity in location of the serotypically unique antigenic determinants of the two M proteins suggested that they may have a common functional domain. This hypothesis was substantiated by the finding that the two M proteins and various chimeras expressed in CV-1 cells by a recombinant vaccinia virus system were able to rescue M gene temperature-sensitive mutants of both VSV serotypes.

Transcription inhibition and other properties of matrix proteins expressed by M genes cloned from measles viruses and diseased human brain tissue.

Ribonucleoprotein (RNP) cores extracted from virions of wild-type (Edmonston strain) measles virus (MV) or obtained from MV-infected cells (cRNP) were shown to be capable of transcribing RNA in vitro but at relatively low efficiency. The tightly bound matrix (M) protein could be effectively removed from virion RNP (vRNP) and from cRNP by exposure to buffers of high ionic strength (0.5 to 1.0 M KCl) but only at pH 8.0 or higher. The vRNP and cRNP cores complexed with M protein exhibited markedly reduced transcriptional activity at increasing concentrations, whereas vRNP and cRNP cores free of M protein exhibited linear and substantially higher transcriptional activity; these data suggest that M protein is the endogenous inhibitor of MV RNP transcription. M-gene cDNA clones derived from three strains of wild-type (wt) MV and 10 clones from mRNAs isolated from the brain tissue of patients who had died from subacute sclerosing panencephalitis (SSPE) and from measles inclusion body encephalitis (MIBE) were recloned in the pTM-1 expression vector driven by the bacteriophage T7 RNA polymerase expressed by a coinfecting vaccinia virus recombinant. All 10 mutant SSPE and MIBE clones expressed in vitro and in vivo M proteins that reacted with monospecific anti-M polyclonal antibody and migrated on polyacrylamide gels to positions identical to or only slightly different from those of the M proteins expressed by wt MV clones. When reconstituted with cRNP cores, the three expressed wt M proteins and 6 of the 10 mutant-expressed M proteins showed equivalent capacity to down-regulate MV transcription. Three of the M proteins from SSPE clones and one from the MIBE clone showed little or no capacity to down-regulate transcription when reconstituted with cRNP cores. The only plausible explanations for loss of transcription inhibition activity by the four SSPE/MIBE M proteins were exceedingly high degrees of hypermutations leading to U-->C transitions and cloning-corrected mutations in the initiator codon (ATG-->ACG) of the four M genes. However, only the hypermutated M protein expressed by the MIBE cDNA clone exhibited virtually no capacity to bind cRNP cores in a reconstitution assay. These experiments provide some preliminary data to support the hypothesis that MV encephalitis may result from certain selective mutations in the M gene.

Viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) has been found to promote assembly and budding of virions as well as down-regulating of VSV transcription. Large quantities of M protein can be produced in insect cells infected with recombinant baculovirus expressing the VSV M gene under control of the polyhedrin promoter. Analysis by pulse-chase experiments and density gradient centrifugation revealed that the [35S]methionine-labeled M protein synthesized in insect cells is released into the extracellular medium in association with lipid vesicles (liposomes). Electron microscopy and immunogold labeling showed that M protein expressed in insect cells induced the formation on plasma membrane of vesicles containing M protein, which are released from the cell surface in the form of liposomes. The baculovirus vector itself or recombinants expressing VSV glycoprotein (G) or nucleocapsid (N) protein did not produce the formation of vesicles in infected cells. The baculovirus-expressed M protein retains biological activity as demonstrated by its capacity to inhibit transcription when reconstituted with VSV nucleocapsids in vitro. These data suggest that M protein has the capacity to associate with the plasma membrane of infected cells and, in so doing, causes evagination of the membrane to form a vesicle which is released from the cell. This observation leads to the postulate, which requires further proof, that the VSV M protein can induce the formation and budding of liposomes from the cell membrane surface.

Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which ostensibly determines the conformational structure of the VSV-New Jersey G protein required for presentation of the major discontinuous epitopes recognized by neutralizing MAbs.

Down-regulation of vesicular stomatitis virus transcription by the matrix protein of influenza virus.

The matrix (M1) protein isolated from influenza A/WSN/33 virus, when reconstituted with ribonucleoprotein (RNP) cores of vesicular stomatitis virus (VSV), resulted in inhibition of VSV transcription in vitro. The presence of endogenous wild-type (wt) or mutant (tsO23) VSV matrix (M) protein on RNP cores did not prevent down-regulation of VSV transcription by reconstituted influenza virus M1 protein. In fact, endogenous VSV wt M protein augmented transcription inhibition by M1 protein reconstituted with RNP/M protein cores, whereas mutant tsO23 M protein endogenous to RNP cores had no effect on down-regulation of VSV transcription by M1 protein. These data suggest that VSV M protein and influenza virus M1 protein recognize two different sites on RNP cores responsible for down-regulation of VSV transcription. Monoclonal antibodies (MAbs) directed to epitope 2 of M1 protein had been previously shown to reverse transcription inhibition by M1 protein on influenza virus RNP cores, but the same epitope 2-specific MAb had little effect on transcription inhibition by M1 protein reconstituted with VSV RNP cores. VSV M protein bears a striking resemblance biologically and genetically to the M1 protein, including, as shown here, their capacity to bind viral RNA. However, the VSV wt M protein exhibited no capacity to down-regulate transcription by influenza virus RNP cores. The significance of these studies is the identification on VSV RNP templates of at least two separate sites for recognition of protein factors that repress VSV transcription.

Effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

The conformational epitopes reactive with neutralizing monoclonal antibodies (MAbs) appear to be clustered at the middle third of the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) and are flanked by two N-linked carbohydrate chains (W. Keil and R.R. Wagner, Virology 170, 392-407, 1989). We report here studies on the effect of glycosylation on the reactivity of VSV-NJ G protein derived from released virions or immunoprecipitated from pulse-labeled cells was not significantly affected in its reactivity with MAbs directed to epitope IV mapped toward the amino-terminus, nor to the centrally located conformational epitopes VI, VIII, and IX. However, there was a 5- to 15-fold decrease in the reactivity with MAb of epitopes VI, VIII, and IX on unglycosylated G protein either isolated from a ribosome-enriched membrane fraction or immunoprecipitated from whole VSV-infected cells labeled for 15 hr in the presence of tunicamycin. In sharp contrast, epitope V and to a somewhat lesser extent epitope VII exhibited decreased reactivity with their respective MAbs when unglycosylated G protein was isolated from released viral particles or from pulse-labeled cells infected with VSV-NJ in the presence of tunicamycin. Enzymatic removal of preformed carbohydrate chains with N-glycanase had little or no effect on the MAb-reactivity of epitopes V and VII, indicating that the carbohydrate chains per se do not influence the antigenic specificity of VSV-NJ G protein. These data suggest that the formation of N-linked carbohydrate chains influences the structure of the VSV-NJ G protein in such a way that epitopes V and VII are shielded from reactivity with their specific MAbs from an early stage of G-protein processing and to a much lesser extent epitopes VI, VIII, and IX at late stages of intracellular processing. These results are compatible with, but do not prove, the hypothesis that N-linked glycosylation plays a key role in promoting the formation and the stability of the disulfide bonds that determine the epitope-specific conformational integrity of the VSV-NJ glycoprotein.

Spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates.

Strains of vesicular stomatitis virus, New Jersey serotype (VSV-NJ), isolated from diseased cattle or swine were examined by genomic RNA sequencing for genetic diversity potentially leading to antigenic variations in their type-specific glycoproteins as determined by reactivity with epitope-specific monoclonal antibodies (MAbs). Seven field isolates recovered in Colorado, New Mexico, Georgia, and Mexico during the widespread 1982-1985 epizootic in the western United States resembled the prototypic 1952 Hazelhurst subtype by partial sequence homology, but amino acid reversions to the 1949 Ogden subtype occurred frequently. When studies were performed with MAbs directed to the Ogden subtype glycoprotein, relatively limited antigenic variation, and only in neutralization epitope VIII, was noted among two of five epizootic isolates from Colorado and New Mexico. However, amino acid differences in the glycoprotein of a 1983 isolate from an enzootic region of Georgia resulted in major antigenic deficiencies in epitopes V, VI, and VII as determined by Western blotting and neutralization of infectivity with epitope-specific MAbs. Quite a few genetic but no antigenic differences were noted in an enzootic 1984 isolate from Mexico, a potential origin of the United States epizootic. Marked or complete loss of epitopes VII, VI, VIII, and V can be traced to spontaneous mutations leading to amino acid substitutions at glycoprotein positions 199, 263, 275, and 317, respectively, in the enzootic Georgia isolate 07/83-GA-P and the epizootic New Mexico isolate 06/85-NM-B. By comparison, closely adjacent amino acid substitutions at glycoprotein positions 210, 268, 277, and 364 occurred in epitope-deficient mutants selected for resistance to neutralization by MAbs specific for epitopes VII, VI, VIII, and V, respectively. Two neutralization epitopes designated X and XI were found to be unique for the G protein of the 1952 Hazelhurst isolate..../52-GA-P. The epitope X-specific MAb H21, in particular, failed to neutralize the infectivity not only of the Ogden subtype..../49-UT-B but also was ineffective against all the 1982-1985 field isolates. The classical 1952 Hazelhurst strain of VSV-NJ is genetically and antigenically quite different from those viruses isolated during the 1982-1985 epizootic.

Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides.

We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M1) protein responsible for down-regulating endogenous transcription of A/WSN/33 influenza virus. A more refined map of the antigenic determinants of the M1 protein was obtained by binding of epitope-specific monoclonal antibodies (MAbs) to chemically cleaved fragments. Epitope 2-specific MAb 289/4 and MAb 7E5 reverse transcription inhibition by M1 protein and react with a 4-kilodalton cyanogen bromide fragment extending from amino acid Gly-129 to Gln-164. Anti-idiotype serum immunoglobulin G prepared in rabbits immunized with MAb 289/4 or MAb 7E5 mimicked the action of M1 protein by inhibiting transcription in vitro of influenza virus ribonucleoprotein cores. This transcription-inhibition activity of anti-MAb 7E5 immunoglobulin G and anti-MAb 289/4 immunoglobulin G could be reversed by MAb 7E5 and MAb 289/4 or could be removed by MAb 7E5-Sepharose affinity chromatography. Transcription of influenza virus ribonucleoprotein was inhibited by one of three synthetic oligopeptides, a nonodecapeptide SP3 with an amino acid sequence corresponding to Pro-90 through Thr-108 of the M1 protein. Of all the structural proteins of influenza virus, only NP and M1 showed strong affinity for binding viral RNA or other extraneous RNAs. The 4-kilodalton cyanogen bromide peptide (Gly-129 to Gln-164), exhibited marked affinity for viral RNA, the binding of which was blocked by epitope 2-specific MAb 7E5 but not by MAbs directed to three other epitopes. Viral RNA also bound strongly to the nonodecapeptide SP3 and rather less well to anti-idiotype anti-MAb 7E5; these latter viral RNA-binding reactions were only slightly blocked by preincubation of anti-MAb 7E5 or SP3 with MAb 7E5. These experiments suggest the presence of at least two RNA-binding sites, which also serve as transcription-inhibition sites, centered around amino acid sequences 80 through 109 (epitope 4?) and 129 through 164 (epitope 2) of the 252 amino acid M1 protein of A/WSN/33 influenza virus. A hydropathy plot of the M1 protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an alpha-helix-forming intervening hydrophobic domain.

Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector.

Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.

Transcription inhibition site on the M protein of vesicular stomatitis virus located by marker rescue of mutant tsO23(III) with M-gene expression vectors.

The matrix (M) protein of vesicular stomatitis virus serves as an endogenous inhibitor of viral transcription, a function missing or deficient in M proteins of temperature-sensitive (ts) mutants assigned to complementation group III. Previous studies with mutant tsO23(III) and vaccinia virus M-gene expression vectors revealed that the temperature-sensitive phenotype is due to a mutation leading to substitution of phenylalanine for leucine at amino acid III, whereas loss of the major antigenic determinant (epitope 1) of the mutant M protein results from the substitution of glutamic acid for the wild-type amino acid glycine at position 21 (Y. Li, L. Luo, R. M. Snyder, and R. R. Wagner, J. Virol. 62:3729-3737, 1988). We demonstrate here that transcription inhibition activity is restored to rescued tsO23 virus only when the rescuing vaccinia virus recombinant expresses M protein with glycine and not glutamic acid at amino acid 21. These experiments indicate the importance of the conformational integrity of the amino-terminal domain in determining the capacity of the vesicular stomatitis virus M protein to down regulate endogenous transcription.

Site-specific mutations in vectors that express antigenic and temperature-sensitive phenotypes of the M gene of vesicular stomatitis virus.

Full-length cDNA copies of mRNAs coding for the matrix (M) proteins of vesicular stomatitis virus and its mutant tsO23(III) were cloned in pBSM13- (BlueScribe). The authenticity of these clones was demonstrated by restriction enzyme mapping, DNA sequencing, and in vitro transcription and translation to identify the two M proteins by Western immunoblotting with epitope-specific monoclonal antibodies. Site-directed mutants were constructed by primer extension of synthetic oligodeoxynucleotides with one or two nucleotide changes to alter the glycine at amino acid 21 of the wild-type (wt) M gene to glutamic acid, alanine, or proline. Similarly, a revertant was created in the M gene of mutant tsO23 by a Glu-21----Gly substitution. A series of wt- and mutant-M-gene chimeras was also constructed to create mutant and revertant clones with Leu----Phe and His----Tyr alterations at amino acids 111 and 227, respectively. We then moved the wt and tsO23 M genes and their site-specific mutants and chimeras cloned in pBSM13- into the eucaryotic expression vector pTF7 directed by the T7 bacteriophage RNA polymerase of the vaccinia virus recombinant vTF1-6,2. Western blot analysis of the M proteins transiently expressed in CV-1 cells by plasmids carrying M genes altered at amino acid 21 revealed that the critical antigenic determinant (epitope 1) is expressed only by the Gly-21 M protein and not by Glu-21, Ala-21, or Pro-21 M proteins. Of particular interest is an apparent conformational change, evidenced by slightly but significantly retarded electrophoretic migration, in plasmid-expressed M proteins with amino acids substituted for glycine at position 21. The glutamic acid at position 21 of tsO23 is not responsible for its temperature-sensitive phenotype, because a tsO23 revertant plasmid with glycine substituted at position 21 fails to rescue tsO23 virus in cells infected at the restrictive temperature; conversely, plasmids expressing wt M protein with substitutions of glutamic acid, alanine, or proline at position 21 are just as effective in marker rescue of tsO23 as is the Gly-21 wt M protein. Marker rescue experiments with wt- and mutant-M-gene chimeras support the hypothesis of K. Morita, R. Vanderoef, and J. Lenard (J. Virol. 61:256-263, 1987) that the temperature-sensitive phenotype of tsO23 is due to a phenylalanine substituted for leucine at amino acid 111, rather than the His-227----Tyr substitution or the Gly-21----Glu substitution, which independently accounts for the loss of epitope 1 in the mutant M protein of tsO23.(ABSTRACT TRUNCATED AT 400 WORDS)